碩士 / 國立高雄海洋科技大學 / 海洋生物技術研究所 / 100 / The chitosanase from Bacillus circulans MH-K1 is classified in glycoside hydrolase family 46. The degree of polymerization of hydrolysates are higher than 2 causing by its endo-splicing pattern (-2)(-1)(1)(2)(3)(4). In order to generate an exo-chitosanase with major product of trimer, the structure relationship between enzyme and substrate were investigated. According to the structures, two strategies were designed—an exo-loop mutagenesis and a saturation mutagenesis at A174/L175.
Firstly, 32 exo-loops with 2-8 amino acids were inserted computationally between Thr180 and Gly182. Five of them were selected and further mutated by site-directed mutagenesis. After over-expression in E. coli BL21(DE3), crude extraction of exo-loop mutants was obtained for chitosan hydrolysis, and the hydrolysates were assayed by TLC. All of the mutants have the ability of endo-type chitosanase activity. However, the hydrolysis products were trended to (GlcN)2 by mutant TMH8 in which the sequence of exo-loop was RPKL.
Secondly, a saturation mutagenesis was performed on residues 174 and 175 for weakening the binding between enzyme and the +2 position of substrate. For high-throughput screening of the mutation library, an in-situ screening was established. It is an efficient screening method that analyzes the hydrolysate of clear zone from chitosanase-detection agar plate by TLC. After in-situ screening of 216 mutants, only 58 mutants with chitosanase activities were obtained. Low colony-count with chitosanase indicated that these two residues are critical for Bacillus chitosanase. After DNA sequencing, 2 higher activity mutants (CSN-II and CSN-TI), 2 lower activity mutants (CSN-LN and CSN-NL), and 3 non-activity mutants (CSN-KT, CSN-HG and CSN-IN) were identified. When cultured in LB medium, most mutated chitosanase were expressed as inclusion bodies, and most of them were resolved by lower induction temperature. According to TLC results, (GlcN)2 of hydrolysate distribution was almost reduced by mutants. Two mutants, CSN-TI and CSN-LN, were selected for their particularly different products. The hydrolysate distribution of wild type, CSN-TI and CSN-LN were analyzed by TLC from 18 to 144 minutes. During the hydrolysis reaction, the main product of wild type, CSN-TI and CSN-LN were (GlcN)2, (GlcN)3 and (GlcN)3, respectively. The end product (after 30 hours) was analyzed by TLC and quantification. The (GlcN)3 / (GlcN)2 ratio of wild type, CSN-TI and CSN-LN were 0.61 ± 0.04、0.94 ± 0.06 and 1.4 ± 0.1, respectively. Besides, (GlcN)4 was almost hydrolysed by wild type; but remaining about 10% by CSN-TI and CSN-LN. It represents that mutagenesis on residues 174 and 175, especially A174T/L175I and A174L/L175N, do weaken the binding of the +2 position of substrate. Based on the mutagenesis of exo-loop and residues corresponding to the +2 position of substrate, more chitosanase with potential hydrolysate can be designed in the near future.
| Identifer | oai:union.ndltd.org:TW/100NKIMT270007 |
| Date | January 2012 |
| Creators | Tsai, Chia-Huang, 蔡嘉煌 |
| Contributors | Cheng, Chih-Yu, 鄭至玉 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 76 |
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